The zinc-finger protein CLAMP promotes gypsy chromatin insulator function In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. We identified a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is depleted, gypsy-dependent enhancer blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis revealed extensive overlap particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, but depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity. Shep RNA-binding capacity is required for antagonism of gypsy chromatin insulator activity Recent studies implicate involvement of RNA-binding proteins and specific transcripts in regulation of insulator activities, but mechanisms by which these factors function remain undefined. The Drosophila RNA-binding protein Shep has been shown to repress gypsy insulator function. We generated a point-mutant Shep disrupted for RNA-binding that consequently fails to antagonize gypsy insulator activities in vivo or to rescue developmental defects of shep mutant neurons. We also identified a sufficient minimal wildtype Shep isoform that antagonizes gypsy insulator activities. These results indicate that Shep insulator antagonism requires its RNA-binding capacity. Our study provides new tools and mechanistic insights to understand insulator regulation. Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. We showed that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, we found specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.